Biology subjects

Hodges, E.

Publications and source records attributed to Hodges, E..

9 recordsLinked to original sources

bioRxiv2024

Temporally discordant chromatin accessibility and DNA demethylation define short and long-term enhancer regulation during cell fate specification

Epigenetic mechanisms govern the transcriptional activity of lineage-specifying enhancers; but recent work challenges the dogma that joint chromatin accessibility and DNA demethylation are prerequisites for transcription. To understand this paradox, we established a highly-resolved timeline of DNA demethylation, chromatin accessibility, and transcription factor occupancy during neural progenitor cell differentiation. We show thousands of enhancers undergo rapid, transient accessibility changes associated with distinct periods of transcription factor expression. However, most DNA methylation changes are unidirectional and delayed relative to chromatin dynamics, creating transiently discordant epigenetic states. Genome-wide detection of 5-hydroxymethylcytosine further revealed active demethylation begins ahead of chromatin and transcription factor activity, while enhancer hypomethylation persists long after these activities have dissipated. We demonstrate that these timepoint specific methylation states predict past, present and future chromatin accessibility using machine learning models. Thus, chromatin and DNA methylation collaborate on different timescales to mediate short and long-term enhancer regulation during cell fate specification.

genomics↗

bioRxiv2024

Human TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, the most severe symptoms of TSC are often neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes, we observed aberrant early neurodevelopment in vitro, including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions, including loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued.

developmental biology↗

bioRxiv2024

Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations

BackgroundRecent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype. ResultsIn this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases. ConclusionsUsing our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

genomics↗

bioRxiv2023

Human gene regulatory evolution is driven by the divergence of regulatory element function in both cis and trans

Gene regulatory divergence between species can result from cis-acting local changes to regulatory element DNA sequences or global trans-acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by challenges in identifying trans-acting changes. We present a comprehensive approach to directly identify cis- and trans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using ATAC-STARR-seq. In addition to thousands of cis changes, we discover an unexpected number (~10,000) of trans changes and show that cis and trans elements exhibit distinct patterns of sequence divergence and function. We further identify differentially expressed transcription factors that underlie >50% of trans differences and trace how cis changes can produce cascades of trans changes. Overall, we find that most divergent elements (67%) experienced changes in both cis and trans, revealing a substantial role for trans divergence--alone and together with cis changes--to regulatory differences between species.

genomics↗

bioRxiv2022

Cross-tissue patterns of DNA hypomethylation reveal genetically distinct histories of cell development

Establishment of DNA methylation (DNAme) patterns is essential for balanced multi-lineage cellular differentiation, but exactly how these patterns drive cellular phenotypes is unclear. While >80% of CpG sites are stably methylated, tens of thousands of discrete CpG loci form hypomethylated regions (HMRs). Because they lack DNAme, HMRs are considered transcriptionally permissive, but not all HMRs actively regulate genes. Unlike promoter HMRs, a subset of non-coding HMRs is cell-type specific and enriched for tissue specific gene regulatory functions. Our data further argues not only that HMR establishment is an important step in enforcing cell identity, but also that complex HMR patterns are functionally instructive to gene regulation. To understand the significance of non-coding HMRs, we systematically dissected HMR patterns across diverse human cell types and developmental timepoints, including embryonic, fetal, and adult tissues. Unsupervised clustering of 102,390 distinct HMRs revealed that levels of HMR specificity reflects a developmental hierarchy supported by enrichment of stage-specific transcription factors and gene ontologies. Using a pseudo-time course of development from embryonic stem cells to adult stem and mature hematopoietic cells, we find that most HMRs observed in differentiated cells (~70-75%) are established at early developmental stages and accumulate as development progresses. HMRs that arise during differentiation frequently (~35%) establish near existing HMRs ([≤] 6kb away), leading to the formation of HMR clusters associated with stronger enhancer activity. Using SNP-based partitioned heritability from GWAS summary statistics across diverse traits and clinical lab values, we discovered that genetic contribution to trait heritability is enriched within HMRs. Moreover, the contribution of heritability to cell-relevant traits increases with both increasing developmental specificity and HMR clustering, supporting the role of distinct HMR subsets in regulating normal cell function. Altogether, our findings reveal that HMRs can predict cellular phenotypes by providing genetically distinct historical records of a cells journey through development. AUTHOR SUMMARYStudies aiming to understand the relationship between DNA methylation patterns and phenotypic outcomes have focused largely on individual differentially methylated regions without consideration of combinatorial changes that drive phenotypes. In non-disease contexts, most of the human genome is stably methylated, except for thousands of discrete DNA hypomethylated regions (HMRs) coinciding with gene regulatory elements. Here, we comprehensively characterize HMR relationships both within and between developmentally diverse cell types to understand the functional significance of complex HMR patterns. We show that levels of HMR specificity across cell-types captures time-point specific branchpoints of development. Our analysis further reveals that HMRs form clusters in proximity to cell identity genes and are associated with stronger gene enhancer activity. This is a wide-spread phenomenon and only a very small subset of HMR clusters is explained by overlapping super-enhancer annotations. Partitioned heritability revealed the functional significance of different HMR patterns linked to specific phenotypic outcomes and indicates a quantitative relationship between HMR patterns and complex trait heritability. Altogether, our findings reveal that HMRs can predict cellular phenotypes by providing genetically distinct historical records of a cells journey through development, ultimately providing novel insights into how DNA hypo-methylation mediates genome function.

genomics↗

bioRxiv2022

Identifying transcription factor-bound gene activators and silencers in the chromatin accessible human genome using ATAC-STARR-seq

Massively parallel reporter assays test the capacity of putative gene regulatory elements to drive transcription on a genome-wide scale. Most gene regulatory activity occurs within accessible chromatin, and recently described methods have combined assays that capture these regions--such as assay for transposase-accessible chromatin using sequencing (ATAC-seq)--with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of accessible DNA (ATAC-STARR-seq). Here, we report a multi-omic approach that quantifies regulatory activity, chromatin accessibility, and transcription factor (TF) occupancy with one assay using ATAC-STARR-seq. Our strategy, including important updates to the ATAC-STARR-seq assay design and workflow, enabled high-resolution testing of [~]50 million unique DNA fragments tiling [~]101,000 accessible chromatin regions in human lymphoblastoid cells. We discovered that 30% of all accessible regions contain an activator, a silencer or both. We demonstrate that activators and silencers represent distinct functional groups that are enriched for unique sets of TF motifs and are marked by specific combinations of histone modifications. Using Tn5 cut-sites retained by the ATAC-STARR library, we performed TF footprinting and stratified these groups by the presence of specific TF footprints that are supported by chromatin immunoprecipitation data. We found that activators and silencers clustered by distinct TF footprint combinations are enriched for distinct gene regulatory pathways, and thus, represent distinct gene regulatory networks of human lymphoblastoid cell function. Altogether, these data highlight the multi-faceted capabilities of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome all from a single DNA fragment source.

genomics↗

bioRxiv2020

In vivo genetic screen identifies a SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia

An enhanced requirement for extracellular nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy for evaluating dependencies in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a unique vulnerability in this disease. In accord with this transport function, we demonstrate that the SLC5A3 dependency reflects a myo-inositol auxotrophy in AML. Importantly, the commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate limiting enzyme for myoinositol biosynthesis, inositol-3-phosphate synthase 1. We used gain- and loss-of-function experiments to demonstrate a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hypermethylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with the presence of IDH1/IDH2 and CEBPA mutations. Collectively, our findings reveal myo-inositol auxotrophy as a novel form of metabolic dysregulation in AML, which is caused by the aberrant silencing of a biosynthetic enzyme. Statement of significanceHere, we show how epigenetic silencing can provoke a nutrient dependency in AML by exploiting a synthetic lethality relationship between biosynthesis and transport of myo-inositol. Blocking the function of this solute carrier may have therapeutic potential in an epigenetically-defined subset of AML.

cancer biology↗

bioRxiv2020

Transcriptional silencing of ALDH2 in acute myeloid leukemia confers a dependency on Fanconi anemia proteins

Hundreds of genes become aberrantly silenced in acute myeloid leukemia (AML), with most of these epigenetic changes being of unknown functional consequence. Here, we demonstrate how gene silencing can lead to an acquired dependency on the DNA repair machinery in AML. We make this observation by profiling the essentiality of the ubiquitin conjugation and ligation machinery in cancer cell lines using domain-focused CRISPR screening, which revealed Fanconi anemia (FA) proteins UBE2T (an E2) and FANCL (an E3) as unique dependencies in AML. We demonstrate that these dependencies are due to a synthetic lethal interaction between FA proteins and Aldehyde Dehydrogenase 2 (ALDH2), which function in parallel pathways to counteract the genotoxic effects of endogenous aldehydes. We provide evidence that DNA hypermethylation and transcriptional silencing of ALDH2 occur in a recurrent manner in human AML patient samples, which is sufficient to confer FA pathway dependency in this disease. Taken together, our study suggests that targeting of the ubiquitination reaction catalyzed by FA proteins can eliminate ALDH2-deficient AML.

cancer biology↗

bioRxiv2019

Neanderthal introgression reintroduced functional alleles lost in the human out of Africa bottleneck

Neanderthal ancestry remains across modern Eurasian genomes, and introgressed sequences influence diverse phenotypes, including immune, skin, and neuropsychiatric diseases. Interpretation of introgressed sequences has focused on alleles derived in the Neanderthal lineage. Here, we demonstrate that Neanderthal introgression also reintroduced thousands of ancestral hominin alleles lost in the Eurasian out of Africa bottleneck. Combining evolutionary simulations, expression quantitative trait loci (eQTL), massively parallel reporter assay (MPRA) data, and in vitro validation, we show that reintroduced alleles (RAs) have different fitness effects than Neanderthal-derived alleles (NDAs) and that some RAs regulate gene expression independent of NDAs. Illustrating the broad potential influence of RAs, we find that over 70% of known phenotype associations with NDAs are equally associated with RAs. Finally, we discover enrichment for RA eQTL activity in several tissues, with strongest enrichment in the brain. In summary, our study reveals that Neanderthal introgression supplied Eurasians with many lost functional variants and demonstrates that RAs must be considered when evaluating the effects of introgression. ONE SENTENCE SUMMARYNeanderthal interbreeding with modern humans restored to Eurasians, hundreds of thousands of ancient alleles that were lost in the out of Africa bottleneck.

evolutionary biology↗